Use of Aromatase Inhibitors in Breast Carcinoma

Home , Aminoglutethimide, Anastrozole, Antiestrogen, Aromatase inhibitor, Fadrozole, Vorozole

Endocrine-Related Cancer (1999) 6 75-92 Use of aromatase inhibitors in breast carcinoma

R J Santen and H A Harvey1

Department of Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA 1Department of Medicine, Penn State College of Medicine, Hershey, Pennsylvania 17033, USA (Requests for offprints should be addressed to R J Santen)

Abstract Aromatase, a cytochrome P-450 enzyme that catalyzes the conversion of androgens to estrogens, is the major mechanism of estrogen synthesis in the post-menopausal woman. We review some of the recent scientific advances which shed light on the biologic significance, physiology, expression and regulation of aromatase in breast tissue. Inhibition of aromatase, the terminal step in estrogen biosynthesis, provides a way of treating hormone-dependent breast cancer in older patients. Aminoglutethimide was the first widely used aromatase inhibitor but had several clinical drawbacks. Newer agents are considerably more selective, more potent, less toxic and easier to use in the clinical setting. This article reviews the clinical data supporting the use of the potent, oral competitive aromatase inhibitors anastrozole, letrozole and vorozole and the irreversible inhibitors 4-OH androstenedione and exemestane. The more potent compounds inhibit both peripheral and intra-tumoral aromatase. We discuss the evidence supporting the notion that aromatase inhibitors lack cross- resistance with antiestrogens and suggest that the newer, more potent compounds may have a particular application in breast cancer treatment in a setting of adaptive hypersensitivity to estrogens. Currently available aromatase inhibitors are safe and effective in the management of hormone- dependent breast cancer in post-menopausal women failing antiestrogen therapy and should now be used before progestational agents. There is abundant evidence to support testing these compounds as first-line hormonal therapy for metastatic breast cancer as well as part of adjuvant regimens in older patients and quite possibly in chemoprevention trials of breast cancer.

Endocrine-Related Cancer (1999) 6 75-92

Introduction concept is that estrogens can be metabolized to catecholestrogens and then to quinones which directly Epithelial cells of the normal breast undergo dramatic damage DNA. These two processes - the estrogen changes during various events in a woman’s life such as receptor-mediated, genomic effects on proliferation and puberty, the follicular and luteal phases of the menstrual the receptor-independent, genotoxic effects of estrogen cycle, pregnancy and menopause. The co-ordinated metabolites - can act either in an additive or synergistic interaction of growth factors and steroid hormones fashion to cause breast cancer (Santen et al. 1999). regulate the proliferation and differentiated function of Breast cancers which arise in patients can be divided epithelial and stromal cells in the normal mammary gland. into two subtypes: those which are dependent upon The key growth factors are insulin-like growth factor-I, hormones for growth and those which grow independently prolactin, insulin, the fibroblast growth factor family of of hormonal stimulation (Santen et al. 1990). In the growth factors and growth hormone, and major steroid hormone-dependent subtype, the role of estrogens as hormones are estradiol, progesterone and testosterone modulators of mitogenesis overrides the influence of other (Frantz & Wilson 1998). factors. These sex steroids stimulate cell proliferation For the process of inducing breast cancer, estrogens directly by increasing the rate of transcription of early appear to play a predominant role. These sex steroids are response genes such as c-myc and indirectly through believed to initiate and promote the process of breast stimulation of growth factors which are produced largely carcinogenesis by enhancing the rate of cell division and in response to estrogenic regulation (Dickson & Lippman reducing time available for DNA repair. An emerging new 1995).

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Based upon the concept that estrogen is the proximate Physiology and regulation of aromatase regulator of cell proliferation, two general strategies were Aromatase is a cytochrome P-450 enzyme which catalyzes developed for treatment of hormone-dependent breast cancer: blockade of estrogen receptor action and inhibition the rate-limiting step in estrogen biosynthesis, the of estradiol biosynthesis. Antiestrogens such as tamoxifen conversion of androgens to estrogens (Simpson et al. bind to the estrogen receptor and interfere with trans- 1997, Sasano & Harada 1998). Two major androgens, cription of estrogen-induced genes involved in regulating androstenedione and testosterone, serve as substrates for aromatase. The aromatase enzyme consists of a complex cell proliferation. Clinical trials showed tamoxifen to be containing a cytochrome P-450 protein as well as the effective in inducing objective tumor regressions and to be flavoprotein NADPH cytochrome P-450 reductase associated with minimal side-effects and toxicity. The (Simpson et al. 1997). The gene coding for the second strategy, blockade of estradiol biosynthesis, was demonstrated to be feasible using the steroidogenesis cytochrome P-450 protein (P-450 AROM) exceeds 70 kb inhibitor, aminoglutethimide, which produced tumor and is the largest of the cytochrome P-450 family regressions equivalent to those observed with tamoxifen (Simpson et al. 1993). The cDNA of the aromatase gene (Santen et al. 1990). However, side-effects from amino- contains 3.4 kb and encodes a polypeptide of 503 amino acids with a molecular weight of 55 kDa. Approximately glutethimide were considerable and its effects on several 30% homology exists with other cytochrome P-450 steroidogenic enzymes required concomitant use of a proteins. Because its overall homology to other members glucocorticoid (Santen et al. 1982). Consequently, of the P-450 superfamily is low, aromatase belongs to a tamoxifen became the preferred, first-line endocrine agent with which to treat advanced breast cancer. However, the separate gene family designated CYP19. clinical efficacy of aminoglutethimde focused attention Recent studies indicate that the transcription of the upon the need to develop more potent, better tolerated, and aromatase gene is highly regulated (Simpson et al. 1989, more specific inhibitors of estrogen biosynthesis. 1993, 1997). The first exon of the aromatase gene is transcribed into aromatase message but not translated into protein. There exist nine alternative first exons which can Inhibition of estradiol biosynthesis initiate the transcription of aromatase. Each of these alternate exons contains upstream DNA sequences which Multiple strategies could be used to inhibit estradiol can either enhance or silence the transcription of arom- biosynthesis as a treatment for estrogen-dependent breast atase. Different tissues utilize specific alternate exons to cancer. Inhibition of several enzymes in the steroidogenic initiate transcription. For example, the placenta utilizes pathway, including cholesterol side-chain cleavage, 3 alternate exon I.1, the testis alternate exon II, adipose beta-ol-dehydrogenase-delta 4-5 isomerase, 17-alpha tissue I.3 and I.4 and brain If. Enhancers which react with hydroxylase, 17-beta hydroxysteroid dehydrogenase, upstream elements of these alternate exons markedly estrone sulfatase, and aromatase, could be used to reduce stimulate the rate of transcription of the aromatase gene. the biosynthesis of estradiol and potentially cause Thus, each tissue can regulate the amount of aromatase hormone-dependent breast tumor regression. An addition- transcribed in a highly specific manner (Simpson et al. al strategy is the use of exogenous glucocorticoid to inhibit 1993). release of adrenocorticotropin (ACTH) and suppress Aromatase expression occurs in many organs, includ- estrogen production. Finally, synthetic progestins such as ing ovary, placenta, hypothalamus, liver, muscle, adipose megestrol acetate and medroxy-progesterone acetate exert tissue, and breast cancer itself. Aromatase catalyzes three glucocorticoid effects and inhibit estradiol synthesis by separate steroid hydroxylations which are involved in the suppressing ACTH. conversion of androstenedione to estrone or testosterone The ideal strategy would be to block the synthesis of to estradiol. The first two give rise to 19-hydroxy and 19- estrogen without inhibiting production of other important aldehyde structures and the third, although still contro- steroids or giving pharmacological amounts of progestins versial, probably also involves the C-19 methyl group with or glucocorticoids. For this reason, blockade of the release of formic acid (Fishman & Hahn 1987). This terminal step in estradiol biosynthesis catalyzed by the enzymatic action results in the saturation of the A-ring of enzyme aromatase is considered a more specific and the steroid molecule to produce an aromatic structure, therefore preferable strategy. Several pharmaceutical hence the term aromatization. companies sought to develop potent aromatase inhibitors In the premenopausal state, the major source of designed to specifically block estrogen biosynthesis with- aromatase and of its substrates is the ovary. However, out altering glucocorticoid and mineralocorticoid syn- extra-glandular aromatization of adrenal substrates in thesis, and without requiring addition of large amounts of peripheral sites such as fat, liver and muscle also progestins or exogenous glucocorticoid. contributes substantially to the estrogen pool in the early

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Figure 1 Diagrammatic representation of the potency of aromatase inhibitors as reflected by the isotopic kinetic method for determining degree of aromatase inhibition. The percent conversion of androstenedione to estrone is measured isotopically, correcting for losses of estrone by giving 14[C] estrone tracer. Values indicated represent percent inhibition of total body aromatase. follicular and late luteal phases of the menstrual cycle. In surrounding the tumors, preadipocyte fibroblasts contain the postmenopausal state, the ovary loses its complement aromatase activity that can be detected by biochemical of aromatase enzyme although it does continue to secrete assay or immunohistochemical staining (Miller & O'Neil androstenedione. The adrenal subsumes the primary role 1987, Santen et al. 1994). Normal breast tissue also of providing substrate for aromatase by directly secreting contains aromatase as documented by immunohisto- testosterone and androstenedione. In addition, dehydro- chemistry, by demonstration of aromatase message, and epiandrosterone and its sulfate are secreted by the adrenal by enzyme assays of cultured cells (Mor et al. 1998, and converted into the aromatase substrates, andros- Brodie et al. 1999). tenedione and testosterone, in peripheral tissues. The The biologic relevance of in situ estrogen production major source of the aromatase enzyme in postmenopausal by aromatase has been demonstrated by xenograft women is peripheral tissues and particularly fat and experiments which compare tumors containing and not muscle. containing aromatase (Yue et al. 1998). Human breast Recent studies identified an additional, important site cancer cells transfected permanently with the aromatase of estrogen production, breast tissue itself. Two-thirds of enzyme are compared with cells transfected with breast carcinomas contain aromatase and synthesize irrelevant DNA. In these experiments, tumors containing biologically significant amounts of estrogen locally in the the transfected aromatase enzyme have higher amounts of tumor (Abul-Hajj et al. 1979, Miller & O’Neil 1987, estrogen and grow faster than those with transfection of Santen et al. 1994). Proof of local estradiol synthesis irrelevant DNA. Further, these experiments show that includes measurement of tumor aromatase activity by local production of estradiol in the tumor is a greater radiometric or product isolation assays, by immuno- source of estrogen than uptake from plasma (Yue et al. histochemistry, by demonstration of aromatase mRNA in 1998). Taken together, these studies support the tissue, and by aromatase enzyme assays performed on importance of in situ estrogen production by breast tumors cells isolated from human tumors and grown in cell and suggest that aromatase inhibitors in patients must be culture. The expression of aromatase is highest in the sufficiently potent to block intra-tumoral aromatase. stromal compartment of breast tumors (Santen et al. 1994) Breast tumor tissue aromatase can be regulated by but is present in epithelial cells as well. In breast tissue several enhancers of aromatase transcription (Simpson et

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Figure 2 Diagrammatic representation of the spectrum of action of first through third generation aromatase inhibitors. With development of newer inhibitors, the spectrum of action narrows. The third generation aromatase inhibitors act exclusively on the aromatase enzyme and do not appear to exert additional effects. al. 1997). Dexamethasone, phorbol esters, cyclic AMP, associated with troublesome side-effects. On the other interleukin 6, and prostaglandins can all stimulate aroma- hand, aminoglutethimide appeared to be quite effective in tase transcription in cultured breast cancer cells and causing tumor regressions in patients with breast cancer. specifically in the stromal components. Interestingly, For this reason, pharmaceutical companies and individual products secreted by epithelial cells in the breast tumors investigators focused upon developing more potent and appear to stimulate aromatase in the stroma and provide a specific inhibitors. Second and third generation inhibitors means for autoregulation of tumor growth through were developed with 10- to 10000-fold greater potency estrogen production. A rather novel means of regulation of than aminoglutethimide and greater specificity (Figs 1 and aromatase levels was also recently described - the 2). The half-lives of the inhibitors increased with synthesis stabilization of degradation of enzyme (Harada et al. of more potent inhibitors. The third generation aromatase 1999). Aromatase inhibitors bind to the active site of the inhibitors are capable of decreasing the levels of circu- enzyme and, through mechanisms not completely lating estrogens to a greater extent than the first and understood, prevent proteolysis of the aromatase protein. second generation inhibitors in postmenopausal women Each of these mechanisms may enhance the amount of with hormone-dependent breast cancer. Hypothetically, aromatase in tumor tissue and increase the need for very these highly potent agents could also reduce levels of potent aromatase inhibitors. intra-tumoral aromatase activity to a greater extent than the earlier inhibitors but this has not yet been examined. Development of aromatase inhibitors The first aromatase inhibitors were discovered nearly 30 Pharmacologic classification of years ago and included aminoglutethimide and testolo- aromatase inhibitors lactone (Santen et al. 1990). Testololactone was not very A convenient classification divides inhibitors into potent as an inhibitor, and aminoglutethimide blocked mechanism based or ‘suicide inhibitors’ (Type 1) and several P-450-mediated enzymatic reactions and was competitive inhibitors (Type II) (Brodie 1993). Suicide

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Figure 3 Inhibition of plasma estrogen levels as assessed by RIA and by an ultrasensitive, recombinant DNA-based bioassay (Jones et al. 1992). Basal estradiol levels are approximately threefold lower when measured by the ultrasensitive assay. During administration of the aromatase inhibitor, levels fall to 0.05-0.07 pmol/l as assessed by the ultrasensitive assay and to 2-5 pg/ml with the standard RIA. *P < 0.01 vs baseline. inhibitors initially compete with natural substrates (i.e. 1981, 1982). Another method involved measurement of androstenedione and testosterone) for binding to the active each estrogen metabolite in urine with calculation of total site of the enzyme. The enzyme, then, specifically acts aromatized product. This technique provided results upon the inhibitor to yield reactive alkylating species similar to those from measurements of urinary estrone or which form covalent bonds at or near the active site of the estradiol (Lipton et al. 1995). Using these plasma or enzyme. Through this mechanism, the enzyme is irrever- urinary methods, each agent appeared to suppress estrogen sibly inactivated. Competitive inhibitors, on the other levels to concentrations approaching the sensitivity of the hand, bind reversibly to the active site of the enzyme and RIAs used. To gain greater specificity and sensitivity, prevent product formation only as long as the inhibitor investigators utilized the isotopic kinetic technique of occupies the catalytic site. Whereas mechanism-based Siiteri et al. to measure total body aromatase (Grodin et al. inhibitors are exclusively steroidal in type, competitive 1973, Santen et al. 1978, Jones et al. 1992, Dowsett et al. inhibitors consist both of steroidal and non-steroidal 1995). This required administration of tritiated andros- compounds (Brodie 1993). tenedione and 14[C]-estrone to patients under steady-state conditions and measurement of radiochemically pure Methods used to demonstrate tritiated estrone and estradiol (Santen et al. 1978). The 14[C]-estrone allowed correction for losses during aromatase inhibition multiple purification steps. Using this technique, the The standard method to study aromatase inhibitors in degree of inhibition with various inhibitors ranged from patients is to measure either plasma or urinary estrogen by 90 to 99%. RIA. Early studies demonstrated 50-80% inhibition of From these observations, it was recognized that more plasma or urinary estrone or estradiol (Santen et al. 1978, sensitive plasma assays of estradiol were needed. One

Downloaded from Bioscientifica.com at 09/24/2021 11:39:59PM via free access Santen and Harvey: Use of aromatase inhibitors in breast carcinoma approach was the use of the plasma estrone sulfate assay produced durable clinical responses in 30-50% of patients since basal levels of this conjugate in postmenopausal (Santen et al. 1990). This approach, however, had several women are tenfold higher than the levels of unconjugated important drawbacks. First, aminoglutethimide was asso- estrone and estradiol (Samojlik et al. 1982, Lonning et al. ciated with troublesome side-effects, including drowsi- 1997). With this measurement, suppression to 85% of ness, skin rash, and ataxia. Secondly, standard doses of basal values was observed with most inhibitors. Finally, an 1000 mg aminoglutethimide daily could also inhibit other ultrasensitive bioassay of plasma estradiol which was 50- cytochrome P-450-mediated steroid hydroxylations, par- to 100-fold more sensitive than RIA was developed ticularly those involving the cholesterol side-chain (Oerter-Klein et al. 1995). Surprisingly, with this assay, cleavage enzymes (Santen et al. 1990, Cocconi 1994). one could demonstrate suppression to levels of estradiol of This non-selectivity for aromatase led to inhibition of the 0.05-0.07 pg/ml, concentrations substantially lower than biosynthesis of cortisol, aldosterone and also of thyroid the 2-5 pg/ml suppressed levels detected by RIA (Fig. 3). hormone. This necessitated co-administration of the As observed with use of other highly sensitive plasma glucocorticoid, hydrocortisone, and in about 5% of hormone assays, for example for luteinizing hormone patients, thyroxine. (LH), follicle-stimulating hormone (FSH), thyrotropin Four randomized, controlled clinical trials compared (TSH), and growth hormone, the levels measured under aminoglutethimide in combination with hydrocortisone basal conditions and during suppression with these assays with tamoxifen in advanced breast cancer. (Smith et al. reveals much lower values than with insensitive RIAs. 1981, Lipton et al. 1982, Alonso-Munoz et al. 1988, Gale This probably reflects the fact that insensitive assays are et al. 1994). The antiestrogen tamoxifen and the inhibitor measuring a substantial fraction of ‘blank’ or non-specific of estrogen biosynthesis, aminoglutethimide/hydrocorti- assay artifact. With the use of highly sensitive assays, this sone produced similar rates of objective disease regression artifactual measurement is eliminated and the actual and duration of response (Santen et al. 1990, Gale et al. values measured are much lower. Thus with the ultra- 1994). Tamoxifen produced many fewer side-effects than sensitive estradiol bioassay, the basal levels in post- did aminoglutethimide/hydrocortisone. Cross-over re- menopausal women average 1-3 pg/ml (vs 5-20 pg/ml sponses to aminoglutethimide/hydrocortisone in patients with RIA) (Oerter-Klein et al. 1995). During development relapsing on tamoxifen were substantial, ranging from 25 of the second and third generation aromatase inhibitors, to 50% and 36% in the largest randomized study (Gale et each of these methods has been used to demonstrate the al. 1994). In marked contrast, patients initially treated with magnitude of suppression of enzymatic activity. For these aminoglutethimide/hydrocortisone responded less fre- measurements, the isotopic kinetic technique is consid- quently when crossed over to tamoxifen (19%) (Gale et al. ered the ‘gold standard’ since it is highly sensitive and 1994). This observation reinforced the concept that the allows comparison among various inhibitors (Fig. 1). antiestrogens be used as first-line agents and the aromatase inhibitors as second- or third-line therapies. With the development of better aromatase inhibitors, First generation aromatase inhibitors aminoglutethimide is now of historical interest only. The first aromatase inhibitor to be widely used in the treatment of metastatic breast cancer in postmenopausal Second generation aromatase inhibitors women was the drug aminoglutethimide (Santen et al. 1978, 1981, 1982, 1990). Isotopic kinetic studies demonstrated a 90-95% inhibition of aromatase activity Fadrozole (Santen et al. 1978). Plasma estrone and estradiol levels Fadrozole (CGS 16949A; 4-(5,6,7,8-tetrahydro- and urinary estrogens fell by 50-80% in response to this imidazo[1,5a]-pyridin-5yl) benzonitrile monohydro- aromatase inhibitor. An additional effect, described by chloride) is a fairly potent inhibitor of aromatase with an Lonning and colleagues, was the acceleration of inhibitory constant (Ki) of 0.19 nM (vs 600 nM for metabolism of estrogen sulfate (Geisler et al. 1997). This aminoglutethimide) (Harvey et al. 1994, Harvey 1996). effect resulted in further lowering of free estrogen levels Cholesterol side-chain cleavage activity is minimal but C- in plasma and in urine. With further study of amino- 11 hydroxylase inhibitory effects are observed in vitro at glutethimide, multiple metabolic effects were demon- high drug concentrations. strated, including inhibition of 11-beta hydroxylase, Initial dose-seeking studies conducted in patients aldosterone synthase, and thyroxine synthesis as well as demonstrated effective aromatase inhibition at doses of induction of enzymes metabolizing synthetic glucocorti- 1.8-4.0 mg daily (Harvey et al. 1994). A phase II study coids and aminoglutethimide itself (Santen et al. 1990). then compared doses of 0.6 mg three times daily, 1 mg When aminoglutethimide was combined with a twice daily, and 2 mg twice daily. Maximal suppression of corticosteroid such as hydrocortisone, the regimen plasma and urinary estrogens occurred at a dose of 1.0 mg

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Table 1 Comparison of third generation aromatase inhibitors with progestin therapy

Megace vs vorozole* Megace vs anastrozole (1 mg) Megace vs letrozole (2.5 mg) Response parameters Megace Vorozole P Megace Anastrozole P Megace Letrozole P

Overall 28.7 26 NS 22.5 26.7 0.02 21.5 25.3 0.15 survival months months months months months months

Objective 7.6% 10.5% NS 7.9% 10.3% NS 16.4% 23.6% 0.04 response rates (CR+PR)

Clinical Not Not 26.1% 35% NS 32% 35% NS benefit reported reported (CR+PR+ stable > 6 months)

Time to 3.6 2.7 NS 5 5 NS 5.5 5.6 0.07 progression months months months months months months

Number in 452 764 551 study

NS, not significant. *Goss (1998). Megace, megestrol acetate twice daily and minimal effects on cortisol secretion were overall survival. In these two trials, responses to megestrol observed. Basal cortisol and ACTH levels were unaffected acetate were somewhat lower than expected from previous and cortisol levels increased appropriately after exog- studies with objective response rates of 11 and 13% enous synthetic ACTH (cortrosyn) administration in all respectively. Randomized patients receiving fadrozole patients. Basal levels of aldosterone also remained stable experienced objective responses of 11 and 16% which did following administration of all three drug doses. There not differ significantly from those with megestrol. Stable were no changes in urinary or plasma sodium or disease for more than 6 months occurred in 25% of potassium, nor in standing blood pressure to suggest a patients receiving fadrozole and 20% taking megestrol clinical state of aldosterone deficiency. However, acetate. Nausea was more frequent for fadrozole than cortrosyn-stimulated aldosterone levels were significantly megestrol acetate in both trials (22 vs 13% and 36% vs blunted at all three doses. (Santen et al. 1991). Based on 11% respectively). In contrast, edema was commoner with several phase II trials, toxicity attributed to this agent was megestrol acetate (21 vs 12% and 19 vs 12%) as was mild and consisted mainly of nausea, anorexia, fatigue, weight gain. and hot flashes. The potency of the compound, its Two trials compared fadrozole with tamoxifen relatively specific effects on aromatase and its lack of (Falkson & Falkson 1996, Thurlimann et al. 1996). In the toxicity suggested that it might provide a major first, 1 mg fadrozole twice daily was compared with 20 mg improvement over aminoglutethimide for treatment of tamoxifen daily in 212 postmenopausal patients with patients with breast cancer. metastatic breast cancer. Response rates to tamoxifen Two large multicenter phase III trials in the USA (27%) and to fadrozole (20%) did not differ significantly comparing fadrozole hydrochloride to megestrol acetate in nor did response durations (20 months vs 15 months). patients who had received only tamoxifen as prior However, tamoxifen achieved a significantly longer time hormonal therapy have now been completed (Buzdar et al. to treatment failure (8.5 months vs 6 months, P<0.05). In 1996b, Trunet et al. 1997). These two studies accrued a the second study, fadrozole was compared with tamoxifen total of 672 patients. Final clinical results showed that as first-line therapy in a randomized, controlled trial there were no significant differences between the two conducted in South Africa. Response rates to tamoxifen treatment arms of the trials with respect to time to were 48% vs 43% with fadrozole (P=not significant). progression, objective response rates, response duration or However, response duration was significantly longer with

Downloaded from Bioscientifica.com at 09/24/2021 11:39:59PM via free access Santen and Harvey: Use of aromatase inhibitors in breast carcinoma tamoxifen (median duration not reached vs 343 days, occurs and androgenic effects can be demonstrated under P<0.009) as was overall survival (34 months for certain circumstances (Brodie et al. 1981). tamoxifen vs 26 months for fadrozole, P<0.046). 4-OHA (Lentaron) has been studied extensively in Taken together, these studies demonstrated that Europe in postmenopausal women with breast cancer. fadrozole may be inferior to tamoxifen in efficacy and no Data from four phase II clinical trials of 4-OHA better tolerated than megestrol acetate. Based upon these demonstrated a 33% objective regression rate of breast findings, the second generation aromatase inhibitor, cancer in postmenopausal patients previously treated with fadrozole, would likely find its place as third-line therapy. multiple endocrine therapies. Toxicity included six Fadrozole has been approved for the treatment of patients with sterile abscesses due to intramuscular advanced breast cancer in postmenopausal women in injections, two of sufficient severity to warrant discon- Japan. This agent is not likely to be further developed in tinuation of therapy. No androgenic effects were observed the United States since both anastrozole and letrozole (Goss et al. 1986). appear to be more potent and more selective aromatase Hoffken et al. (1990) conducted a large trial of 4-OHA inhibitors. in postmenopausal women. Patients initially received 500 mg intramuscularly every two weeks for 6 weeks and then Careful analysis of the fadrozole/megestrol acetate 250 mg every 2 weeks thereafter. Plasma estradiol levels trials raises the concern that responses to endocrine fell from baseline values of 10-11 pg/ml to levels of therapies appeared to be less frequent than observed in approximately 4 pg/ml for up to 7 months of therapy. The prior studies. For example, the randomized comparison of drug appeared specific since no reduction of cortisol or the first generation aromatase inhibitor, aminoglute- symptoms of cortisol deficiency were observed. Of 86 thimide, with surgical adrenalectomy demonstrated evaluable patients, there were 2 complete and 19 partial responses of 40-50% in patents previously treated with remissions (24%) and 26 with disease stabilization (30%). tamoxifen (Santen et al. 1981). Other studies with Side-effects included minor systemic symptoms in 11% megestrol acetate as second-line therapy demonstrated (hot flashes, constipation, alopecia, and pruritus) and local responses ranging from 30 to 50%. Several possibilities symptoms in 8% (pruritus, local pain, and erythema). could explain the low response rates. In recent studies, These side-effects resulted in discontinuation of therapy in more stringent criteria have been used than in previous only 2% of patients. Phase III trials are now ongoing to trials. For example, recalcification of mixed lytic/blastic compare this inhibitor with standard endocrine therapies. metastases were previously considered objective evidence In general, 4-OHA is better tolerated than amino- of partial responses. Such lesions are now considered non- glutethimide. This agent is not available in the USA. assessable, non-measurable disease. External review of Studies of the degree of aromatase inhibition using cases probably also increases the stringency of assess- isotopic kinetic techniques demonstrate that 4-OHA is not ment. It should be noted that in a previous study as effective as the third generation inhibitors in blocking comparing tamoxifen alone vs tamoxifen and fluoxy- estrogen production (Fig. 1). For this reason and because mestrone, the objective response rate for tamoxifen alone it must be injected i.m. it is unlikely that this agent will was only 10% (Swain et al. 1988). These considerations compete successfully with the newer inhibitors. lead to the conclusion that one can only compare new agents with established ones such as tamoxifen and assess the relative differences between them. It is inappropriate Exemestane to compare the percent of objective responses to those Another steroidal aromatase inhibitor under active observed in historical controls. investigation is exemestane. Exemestane (6-methylene- androsta-1,4-diene-3,17-dione) is an irreversible (Type I or mechanism-based) aromatase inhibitor (Evans et al. 4-Hydroxyandrostenedione (4-OHA) 1992, Thurlimann et al. 1997, Lonning 1998). Its Ki for Formestane (Lentaron; 4-OHA; 4-hydroxyandrost-4-ene- competitive inhibition is 10.2 nmol/l and for irreversible 3,17-dione) is a structural analog of androstenedione and inactivation is K (intact) of 26 nmol/l. Single dose is thus a highly specific aromatase inhibitor (Lonning administration reveals a major reduction of plasma 1998). It was the first steroidal suicide-type (Type l) estrogens with this compound (Lonning 1998). A dose of aromatase inhibitor to enter clinical trials and is now 25 mg daily inhibited aromatase activity as documented commercially available in Europe. Using the in vitro by the isotope kinetic technique by 97.9%. Thurlimann et placental aromatase assay system, 4-OHA was shown to al. (1997) reported an objective response (complete be 60-fold more potent than aminoglutethimide (Ki=4.1 response (CR) and partial response (PR)) in 12% and 33% µM). Extensive studies revealed no estrogenic, anti- of patients expressing primary or secondary resistance to estrogenic, or antiandrogenic properties (Brodie & Wing aminoglutethimide. Other studies are ongoing but not yet 1987). However, transformation to 4-hydroxytestosterone completed.

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Table 2 Dose response studies with third generation aromatase inhibitors

Anastrazole Letrozole Response parameters 1 mg 10 mg P 0.5 mg 2.5 mg P

Overall 26.7 Not NS 21 months* 28 months 0.04 survival reported 21.5 months** 25.3 months 0.03

Objective 10.3% 8.9% NS 16.7% 19.5% NS response 12.8% 23.6% 0.004 rates (CR+PR)

Clinical benefit 35% 32% NS 32.8% 36.3% NS (CR+PR+ 27% 35% NS stable >6 months)

Time to 5 months 5 months NS 3.3 months 3.4 months NS progression 5.1 months 5.6 months 0.02

Number in 764 555* study 551**

*Gershanovich et al. (1998); **Dombernowsky et al. (1998).

Third generation aromatase inhibitors was progressing following therapy with tamoxifen given either in the adjuvant setting or as first-line endocrine therapy for metastatic disease. Patients in the three arms Anastrozole of the trial had similar prognostic characteristics including Anastrozole (Arimidex; ICI-D1033; 2,2'-[5-(1H-1,2,4- age, estrogen receptor status, disease-free interval, and triazol-1-ylmethyl)-1,3-phenylene]bis(2-methyl-propio- sites of metastases. Results from these important trials nonitrile) is a potent and selective benzyltriazole showed similar overall response rates to either dose of derivative (Buzdar et al. 1996a, 1997, Buzdar 1998). At a anastrozole or to megestrol acetate. No statistically concentration of 15 nmol/l this compound inhibits significant dose-response differences were observed aromatase activity by 50%. In rodents, maximal hormonal between the 1 and the 10 mg daily dosage. The rates of suppression is achieved with an oral dose of 0.1 mg/kg. overall objective response of 10.3% and 8.9% were also Activity is assessed by examining the degree of inhibition surprisingly low, probably for reasons discussed above. of ovulation and of androstenedione-induced uterine Overall responses including complete and partial object- hypertrophy. Studies conducted in monkeys demonstrate 2 ive response rates and stabilization of disease of greater similar inhibitory potency when expressed on a mg/m than 6 months averaged 35%. It should be noted that recent basis and assessed by measurement of plasma estradiol. studies have demonstrated that disease stabilization for Studies in women demonstrated suppression of plasma greater than 6 months is a meaningful clinical parameter estrogen to levels approaching assay sensitivity (Kleeburg since patients experiencing this response survive equally et al. 1997). Anastrazole produces no effects on as long as patients undergoing partial objective response aldosterone, cortisol, or thyroxine synthesis (Kleeburg et (Howell et al. 1998). Patients with complete or partial al. 1997). The estimated elimination half-life in humans is objective responses or stable disease survive longer than 32.2 h. those with disease progression. Anastrozole was the first aromatase inhibitor to be In initial reports, the third generation aromatase approved in the USA for the management of advanced inhibitor, anastrozole, was considered superior to meges- breast carcinoma in postmenopausal women. This terol acetate because it was better tolerated. It was approval was based on results of two pivotal trials that associated with less undesirable weight gain, dyspnea, and together accrued a total of 764 patients randomized to fewer thromboembolic events when compared with receive either anastrozole (1 mg p.o./day) or anastrozole megestrol acetate (Buzdar et al. 1996a, 1997). Since there (10 mg/day) or megestrol acetate 40 mg (q/day) (Buzdar were no differences between the two doses of anastrozole, et al. 1996a). These patients had metastatic disease that the drug was approved at a dose of 1 mg daily.

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Table 3 Comparison of first with third generation aromatase inhibitors

AG/HC vs vorozole AG/HC vs letrozole Response parameters AG/HC Vorozole P AG/HC Letrozole P

Overall 21.7 months 25.7 months NS 20 months 28 months 0.002 survival

Objective 18% 23% 0.085 12.4% 16.7% 0.06 response (CR+PR)

Clinical benefit 37 % 47% 0.017 29.4% 36.3% Not (CR+PR+ reported stable > 6 months)

Time to 6.0 months 6.7 months NS 3.2 months 3.4 months 0.008 progression

Number in 456 555 study

AG, amionoglutethimide; HC, hydrocortisone. *Goss (1998). **Gershanovich et al. (1998).

Surprisingly, with further maturity of this trial, higher than the concentration required to cause a 50% anastrozole (1 or 10mg daily) conferred a survival advan- inhibition of the aromatase enzyme, letrozole does not tage compared with the progestin (median of 26.7 months significantly suppress either aldosterone or corticosterone vs 22.5 months) (Buzdar et al. 1997) (Table 1). The 2-year in rats. Letrozole also causes significant regression of survival was 56.1% for the group of patients receiving anastrozole (1 mg) compared with 46.3% for patients treated with megestrol acetate. The demonstration that Table 4 Comparison of third generation aromatase inhibitors anastrozole has superior efficacy with respect to overall survival and reduced side-effects vs megestrol acetate Response would suggest that the aromatase inhibitor be used as parameters Vorozole Anastrozole Letrozole second-line therapy in preference to megestrol acetate. Overall 25.7 months 26.7 months 28 months* survival 25.3 months** Letrozole The second aromatase inhibitor to gain approval in the Objective 10.5% 10.3% 19.5%* United States with the indication for management of response 23.6%** postmenopausal women with metastatic breast cancer was rates letrozole (Femara) (Dombernowsky et al. 1998, (CR+PR) Gershanovich et al. 1998). Letrozole (4,4'-(1H-1,2,4- Clinical 47% 35% 36.3%* triazol-1-yl-methylene)-bis-benzonitrile) is also a potent benefit 35%** non-steroidal competitive aromatase inhibitor. This agent (CR+PR+ possesses considerable selectivity for aromatase. In stable > 6 preclinical studies, for example, letrozole caused inhibi- months) tion of aldosterone production in vitro only at concentrations 10000 times higher than those required for inhibition Time to 2.7 months 5 months 3.4 months* of estrogen production. Letrozole is a highly potent and progression 5.6 months** selective aromatase inhibitor. When administered orally to Number in 452 764 555* adult female rats at a dose of 1 mg/kg per day for 14 days, study 551** letrozole decreases uterine weight to that observed after a surgical ovariectomy. At doses greater than 1000 times *Gershanovich et al. (1998), **Dombernowsky et al. (1998).

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Dimethylbenzanthracene (DMBA)-induced rat mammary there was a statistically significant improvement in overall tumors (Schiweck et al. 1993). survival for the patients receiving letrozole. Moreover, Clinical studies in normal healthy volunteers as well letrozole produced less somnolence and skin rash. The as dose-seeking phase I trials in postmenopausal women results of these large, well-done, randomized trials suggest with advanced breast cancer showed that letrozole in a that the side-effect profile and the dosing schedules of both dose as little as 0.25 mg p.o. daily caused maximal anastrozole and letrozole are superior to megestrol acetate suppression of plasma and urinary estrogens. A highly and aminoglutethimide. sensitive recombinant DNA-based estradiol bioassay was used to assess estradiol levels in one of these studies Vorozole (Oerter-Klein et al. 1995). The levels of estradiol were This agent is another third generation, non-steroidal, oral decreased by 95% to levels of 0.05-0.07 pmol/l as detected aromatase inhibitor which is highly potent and specific for by this assay (Fig. 3). This observation underscores the aromatase (Goss 1998). Nearly all of the aromatase limitation of standard RIAs for detection of estradiol inhibitor activity resides in the dextroenantiomer levels in patients given highly potent aromatase inhibitors. (R083842). Clinical efficacy appears to be similar to that Additional studies established the fact that letrozole of anastrozole and letrozole (Tables 1 and 2). Vorozol was quite selective for the inhibition of aromatase since, appears to be superior to aminoglutethimide/hydrocorti- over a wide dose range, there were no significant changes sone with respect to clinical benefit (i.e. complete and in the levels of gonadotropins, ACTH, cortisol, partial objective regression plus stabilization of disease aldosterone, or TSH (Demers et al. 1993, Dowsett et al. for greater than 6 months) (Table 3). Its efficacy did not 1995). Early trials of letrozole in heavily pretreated post- differ significantly from that of megestrol acetate although menopausal women with metastatic breast cancer it was associated with fewer side-effects. Because of the demonstrated both clinical efficacy and lack of significant proven efficacy and prior approval of anastrozole and toxicity (Iveson et al. 1993). letrozole, further clinical development of vorozole has Approval of this agent was based on the results of two recently been abandoned and full description of its clinical large, multi-center, randomized trials similar in design to properties is referenced but not detailed. the studies involving anastrozole (Dombernowsky et al. 1998, Gershanovich et al. 1998). In a pivotal trial, 555 postmenopausal women with metastatic breast carcinoma Comparison of potency of progressing after treatment with tamoxifen were random- aromatase inhibitors ized to receive either letrozole (0.5 mg daily), letrozole The relative potencies of aromatase inhibitors can be (2.5 mg daily) or standard doses of megestrol acetate determined in vitro and characterized as inhibitory (Tables 1 and 2). The women in the three treatment groups constants (i.e. Ki ) or as the concentration that inhibits were comparable in all respects. The two doses of aromatase by 50%. However, these measurements do not letrozole caused similar prompt and profound suppression provide information which can be extrapolated to patients. of plasma and urinary estrogens (Demers et al. 1993). In addition to Ki, drug half-life and amount of drug that Letrozole (2.5 mg) yielded an overall response rate can be given safely contribute substantially to degree of (complete and partial tumor regression and disease inhibition achievable in patients. Consequently, the most stabilization for greater than 6 months) of 36% and 35% useful comparator of potency among agents is the compared with 27% and 33% for letrozole (0.5 mg) and measurement of degree of aromatase inhibition in women 32% for megestrol acetate. However, the median duration with breast cancer. This requires highly sensitive and of response for letrozole (2.5 mg) was 33 months specific means of measuring aromatase inhibition. Plasma compared with 18 months for both megestrol acetate and RIA techniques are not sufficiently sensitive to precisely the lower dose of letrozole. Similarly, there was a trend in quantitate degree of suppression and the ultrasensitive time to tumor progression and survival that favors the 2.5 estradiol bioassay has not been used to compare inhibitors. mg letrozole dose. The isotopic kinetic technique for quantitating total In a second and similar study involving 555 body aromatase activity serves then as the best method to postmenopausal patients with advanced breast cancer compare the potency of various inhibitors in patients progressing after tamoxifen therapy, letrozole was com- (Grodin et al. 1973, Santen et al. 1978, Dowsett et al. pared with aminoglutethimide (250 mg bid.) and hydro- 1985, Jones et al. 1992). Lonning and others (Jones et al. cortisone (Table 3). Letrozole (2.5 mg daily) produced an 1992) have compared a number of these agents and objective response rate of 17% vs 12% for reviewed published studies of others. With this method- aminoglutethimide (Gershanovich et al. 1998). The ology, 4-OHA inhibits aromatase by 92%, fadrozole by median response duration was 23 months for letrozole 93%, examestane by 97.9%, anastrozole by 93%, vorozole compared with 15 months for aminoglutethimide and by 98%, and letrozole by 99%. (Fig. 1). It is not clear

Downloaded from Bioscientifica.com at 09/24/2021 11:39:59PM via free access Santen and Harvey: Use of aromatase inhibitors in breast carcinoma whether the aromatase activity remaining during therapy megestrol acetate. Side-effects reported for letrozole and is biologically important. Most biologic systems operate anastrozole were low grade in severity including mild on a log dose-response basis. Since residual aromatase headache, nausea, diarrhea, and hot flashes and were activity is 8% with 4-OHA and only 1% with letrozole, infrequent. Significantly, letrozole and anastrozole were these differences could have biologic relevance. associated with less weight gain, dyspnea, thromboembolic events, and vaginal bleeding when compared with Summary of conclusions from large megestrol acetate (Buzdar et al. 1996a, 1997, Buzdar 1998, Dombernowsky et al. 1998). clinical trials with third generation aromatase inhibitors Both of these aromatase inhibitors, anastrozole and letrozole, are highly potent, specific, and well tolerated. It These studies allow answers to three important questions. is probable that in clinical practice, either of these agents (1) Do higher doses of third generation aromatase inhib- will now replace megestrol acetate or other progestins as itors produce greater clinical effects than do lower doses? second-line therapy after tamoxifen in postmenopausal (2) Do third generation inhibitors produce greater clinical women with metastatic breast carcinoma. benefit than the first generation aromatase inhibitor aminoglutethimide? (3) Do the third generation inhibitors produce greater clinical benefit than does megestrol Relative efficacy of third acetate? A fourth question, ‘Which is the most effective generation inhibitors third generation aromatase inhibitor?’ cannot be answered until head to head comparisons between agents are made. Table 4 compares several parameters observed with the Relative efficacy based upon results among very large, but various third generation inhibitors. Overall survival is non-randomized trials cannot be validly interpreted but quite similar with each agent and ranges from 25.3 months provide trends to be tested in future studies. to 28 months. Objective response rates on the other hand With respect to the dose-response question (question appeared somewhat higher with letrozole (19.5 and 1), two large studies clearly demonstrated that 2.5 mg 23.6%) than with vorozole (10.5%) and anastrozole letrozole is more effective than the 0.5 mg dose. In (10.3%). The percent of patients experiencing clinical contrast, no differences were demonstrated when compar- benefit (i.e. objective response plus stabilization of ing 1 with 10 mg anastrozole daily (Table 2). With respect disease for greater than 6 months) appeared similar for to the superiority of third over first generation inhibitors each therapeutic modality and ranged from 47% with (question 2), letrozole, at the 2.5 mg dosage produced vorozole to 35% with anastrozole to 36.3 and 35% with significantly better responses than did aminoglutethimide letrozole. Time to progression appeared the shortest with with respect to duration of response, time to progression vorozole (2.7 months) and somewhat longer but similar and time to treatment failure (Table 3). The percent with anastrozole (5 months) as with letrozole (3.4-5.6 objective response rates was also greater with letrozole months). Head to head comparisons are now required to than with aminoglutethimide and the rate of side-effects determine if the somewhat longer durations of response was less than with the first generation aromatase inhibitor. and objective response rates observed with letrozole Finally, with respect to the superiority of third generation represent statistically significant differences. inhibitors to other agents (question 3), anastrozole was clearly superior to megestrol with respect to overall patient survival (Table 1). Letrozole was also superior in Comparisons of aromatase inhibitors clinical efficacy to megestrol with respect to percent with antiestrogens as first-line objective responses, time to progression and time to endocrine therapy treatment failure. The overall duration of survival was not significantly different but the trend favored 2.5 mg Prior studies demonstrated that tamoxifen and letrozole (P=0.10). With greater maturity of these studies, aminoglutethimde/hydrocortisone when compared in four differences in overall survival could emerge. It should be randomized, controlled, double-blind studies showed noted that earlier studies (as cited above) suggested equal similar efficacy in patients with advanced beast cancer efficacy of anastrozole and megestrol but updated data (Smith et al. 1981, Lipton et al. 1982, Alonso-Munoz et demonstrate a clear enhancement of overall survival al. 1988, Gale et al. 1994). Since letrozole has been shown imparted by anastrozole when compared with megestrol. superior to aminoglutethimide in a direct comparative Vorozole, on the other hand, did not differ from megestrol trial, it appears logical to determine whether the third with respect to any parameter reflecting efficacy. generation aromatase inhibitors are superior to tamoxifen Each of these trials demonstrated that the third genera- as first-line therapy. These trials are now ongoing but no tion aromatase inhibitors were better tolerated than results are as yet available.

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Comparison of antiestrogens with third approaches. More recently, however, the widespread use generation aromatase inhibitors in the of tamoxifen as adjuvant therapy, frequently administered adjuvant setting for long periods of time, presents the clinician with a new therapeutic dilemma. A practical approach is to rely on Trials are now ongoing to determine the efficacy of tamoxifen as first-line therapy for patients with metastatic aromatase inhibitors vs tamoxifen vs the combination of disease who have not received this agent in the adjuvant antiestrogen and aromatase inhibitor. The largest trial is setting or have discontinued tamoxifen for a period of termed the ATAC trial (anastrozole alone versus greater than 1 year. For other patients who are still tamoxifen alone and in combination - i.e. anastrozole plus candidates for hormone therapy, the major choices at tamoxifen). An anticipated 20000 patients will be enrolled present are between progestins such as megestrol acetate in this trial and currently 500 per month are being entered or medroxyprogesterone acetate and aromatase inhibitors. in 200 centers worldwide. This and other similar trials Given their equal or greater efficacy and better tolerability, should establish the relative efficacies of these two aromatase inhibitors should now replace progestins as therapeutic strategies. second-line hormonal therapy for metastatic breast cancer. Secondary issues in these trials are the differential It has been speculated that the determination of aroma- actions of the antiestrogens and aromatase inhibitors on tase content of a particular tumor by either biochemical non-breast tissues. Tamoxifen acts as an estrogen agonist measurement or immunohistochemistry might aid in on uterus and increases the incidence of uterine cancer selecting patients who are likely to respond to therapy with whereas the aromatase inhibitors would be expected to aromatase inhibitors (Bezwoda et al. 1987). Preliminary reduce estrogenic stimulation on the uterus. The beneficial evidence also suggests that breast cancers that overexpress effects of tamoxifen on bone and potentially on the the Her-2/neu protein may be relatively resistant to cardiovascular system differ from the potential of the hormonal therapies including tamoxifen and aromatase aromatase inhibitors to accelerate the process of bone inhibitors (Leltzel et al. 1995, Yamaguchi et al. 1997). If resorption and the incidence of cardiovascular disease. confirmed, this information together with other consid- Subprojects within the ATAC trial are examining these erations might well assist the clinician in better selecting issues in detail. patients for these forms of therapy.

Selection of patients for aromatase Premenopausal patients inhibition therapy Considerations of efficacy, cost, toxicity, and ease of Endocrine therapy is usually offered to patients with administration also dictate the choice of endocrine therapy metastatic disease who have receptor positive (estrogen in premenopausal patients. Based on these considerations, receptor positive and progesterone receptor positive) or first-line therapy would include either tamoxifen or receptor unknown disease (Santen et al. 1990). In addition oophorectomy. Effective castration can be accomplished to the level of receptors, clinical features that might either by surgery, pelvic irradiation, or the use of suggest a favorable response include a long disease-free luteinizing hormone-releasing hormone analogs. In hu- interval after initial surgery or the presence of nodal, soft mans, the premenopausal ovary has generally been tissue, bone, pleural or nodular lung metastases. Patients considered to be resistant to blockade of estrogen with central nervous system involvement, extensive liver production by aromatase inhibitors since any lowering of disease, lymphangitic spread of tumor in the lungs or plasma estrogens would lead to reflex increases in both rapidly progressing and life-threatening disease are not FSH and LH (Santen et al. 1980). These increased ordinarily considered candidates for hormonal therapy. gonadotropin levels would then induce increased ovarian Considerable clinical experience and data from the litera- production of estradiol and of androstenedione, the major ture suggest that most endocrine therapies with the substrate for aromatase action. On the other hand, the possible exceptions of androgens and glucocorticoids are activity of the very potent third generation inhibitors equally effective. makes it possible that these compounds might also inhibit The decision to choose one endocrine therapy over ovarian steroidogenesis and therefore may be of value in another depends upon the menopausal status of the patient, the treatment of breast cancer in premenopausal women. considerations of efficacy, ease of administration, cost, However, there are no clinical studies demonstrating and side-effects. Historically, of all the endocrine thera- complete ovarian blockade with aromatase inhibitors. One pies, tamoxifen was associated with the fewest side- preliminary study showed no effective inhibition effects. This aspect still favors tamoxifen as the endocrine (Yamaguchi 1999), consequently, the use of aromatase therapy of first choice. Aromatase inhibitors are then inhibitors should be restricted to the treatment of breast considered either as second- or third-line endocrine cancer either in postmenopausal patients or in premeno-

Downloaded from Bioscientifica.com at 09/24/2021 11:39:59PM via free access Santen and Harvey: Use of aromatase inhibitors in breast carcinoma pausal women whose ovaries have been rendered non- to explain why women appear to be benefited to a greater functional by use of gonadotropin-releasing hormone extent with 5 than with 10 years of tamoxifen in the agonists or by surgical or radiation ablation. adjuvant setting. Adaptation to tamoxifen, occurring between 5 and 10 years of exposure to this agent, might allow tamoxifen to ultimately become a stimulator of Mechanisms for lack of cross resistance growth of the remaining micrometastases (Santen 1997). of aromatase inhibitors and antiestrogens Logic would suggest that inhibitors of estrogen action, Adaptive hypersensitivity hypothesis such as tamoxifen, would be completely cross resistant with agents designed to block estrogen synthesis. How- Another possible explanation for secondary responses to ever, early studies demonstrated that sequential responses aromatase inhibitors following exposure to tamoxifen is to inhibitors of estrogen biosynthesis commonly occurred the development of adaptive hypersensitivity to estradiol. in patients initially responding to and then relapsing after This phenomenon was initially suggested by clinical treatment with the antiestrogen, tamoxifen. For example, observations demonstrating sequential tumor regressions 25-50% of patients initially responding to tamoxifen and in women undergoing oophorectomy followed by then relapsing experienced secondary tumor regressions in exposure to an aromatase inhibitor. Oophorectomy response to the aromatase inhibitor aminoglutethimide in reduces estradiol levels from approximately 200 pg/ml combination with hydrocortisone. (premenopausal levels) to 5-10 pg/ml (post-oophorectomy concentrations) resulting in tumor regression. The cancer then begins to regrow in the presence of these low Potential explanations for lack of cross estradiol levels but undergoes further regression when resistance among hormonal therapies aromatase inhibitors lower levels further to 0.05-0.07 One potential explanation for the lack of cross resistance pmol/l. These observations are best explained by the between antiestrogens and aromatase inhibitors was raised hypothesis that long-term deprivation of estradiol can by observations made during further study of the actions induce an adaptive sensitization of the tumor to estradiol. of the antiestrogens. A variety of data examining the One could consider this analogous to Cannon's law of effects of antiestrogens on various organs and in various denervation hypersensitivity whereby estradiol depriva- species demonstrated that estrogen receptor antagonists tion causes hypersensitivity to estradiol. exert both hormone agonistic and antagonistic actions, We tested the estradiol hypersensitivity hypothesis depending upon the tissue studied (Santen 1997). For directly in an in vitro cell culture system (Masamura et al. example, tamoxifen acts as a potent estrogen on bone, 1995). Breast cancer cells were deprived of estradiol over liver, pituitary, and uterus while exerting antiestrogenic several months in culture by growing them in media effects on breast. The various responses to antiestrogens stripped of estradiol by treatment with charcoal. This could be modulated by adaptive mechanisms such as, for period of estrogen deprivation induced a four log example, increased production of cyclic AMP or activa- enhancement in sensitivity to the cell proliferative effects tion of the protein kinases A and C pathways (Santen of estradiol. The hypersensitivity phenomenon could be 1997). Observations in xenograft models of human breast reversed by re-exposure of cells to estradiol, suggesting cancer were particularly striking with respect to this adaptive mechanisms rather than selection of hyper- adaptive process. Initial exposure to tamoxifen caused sensitive clones of cells. tumor regression but prolonged exposure allowed the Long-term exposure to tamoxifen might also result in tumor to adapt such that tamoxifen shifted from exerting development of hypersensitivity to estradiol. Under these estrogen-antagonistic to estrogen-agonistic effects. Re- circumstances, a marked reduction of estradiol synthesis transplant of the xenografts into additional animals with an aromatase inhibitor would result in tumor allowed demonstration that tamoxifen stimulated these regression. Taken together, these observations suggest that tumors to grow and that the pure antiestrogen, ICI breast cancer cells adapt to the conditions of ambient 182,782, could antagonize this estrogenic effect (Gottardis hormonal exposure, either to tamoxifen or to estrogen & Jordan 1988). deprivation. This adaptive process provides a plausible These observations led to the hypothesis that, in explanation for the sequential responses to various patients, breast tumors initially responding to tamoxifen hormonal therapies observed clinically in women with but then regrowing had also undergone adaptation. Such breast cancer. tumors might then respond secondarily to agents such as Development of adaptive hypersensitivity has the aromatase inhibitors which would lower estrogen practical implications for the use of aromatase inhibitors. levels but not be expected to exert estrogen-agonistic If cells in culture can respond to 10 fM concentration of actions. The hypothesis of adaptation has also been used estradiol, nearly complete inhibition of aromatase may be

Downloaded from Bioscientifica.com at 09/24/2021 11:39:59PM via free access Endocrine-Related Cancer (1999) 6 75-92 necessary to produce most effective anti-tumor therapy. ment of a breast tumor requires further study. Greater Even the most potent inhibitors available now allow 1% understanding of the biologic interaction of these factors residual aromatase activity. It is not clear whether the could lead, for example, to the development of new inhibitors block aromatase in breast tumor tissue itself to therapeutic strategies. the same degree. These concepts are of interest when considering the dose-response differences between 0.5 and 2.5 mg letrazole daily. Perhaps even more potent Use of aromatase inhibitors for breast aromatase inhibitors could produce even greater clinical cancer prevention effects. This possibility is not supported by the lack of Estrogens are considered carcinogenic for the breast dose-response differences detected between 1 and 10 mg through the ability to increase the rate of cellular anastrazole per day but perhaps deserves further explor- proliferation and consequently to increase the number of ation. genetic mutations which are proportional to the number of cell divisions (Santen et al. 1999). In addition, the increased rate of cell proliferation could reduce the time Future perspectives required for DNA repair. This is the commonly accepted As discussed above, several new potent and highly mechanism of estradiol-induced carcinogenesis. An addi- specific aromatase inhibitors are now available for the tional mechanism has been proposed which involves the treatment of breast cancer. They offer several distinct metabolism of estradiol to 4-hydroxyestradiol and then to advantages over some older forms of endocrine therapy the 3,4 estradiol quinone. This compound can bind co- including a well-understood mechanism of action, good valently to guanine or adenine and result in depurination toxicity profile, convenient dosing schedules, and the of that segment of DNA. Upon replication, these absence of estrogen effects on the endometrium. On the depurinated sites preferentially undergo point mutations. other hand, their long-term effects on bone mineral density This process could act in an additive or synergistic fashion and serum lipids are unknown (Harvey 1996). with the effect of estrogen to increase cell proliferation. New clinical trials with these promising agents are It has been postulated that antiestrogens might prevent either underway or are planned in order to address several breast cancer by blocking the cell-proliferative effects of questions including their role in the treatment of estrogens. The aromatase inhibitors might prevent breast premenopausal women as discussed above. Although cancer by two mechanisms: reduction of cell proliferation presently approved only as second-line therapies after by inhibition of estrogen levels and prevention of tamoxifen failure, aromatase inhibitors are now being genotoxic metabolite formation by lowering tissue levels tested as first-line endocrine treatment for metastatic of estrogen. Coombes et al. (1991) have reported that 4- breast cancer in direct comparison to antiestrogens. OHA prevents Nitrosomethylureal (NMU)-induced rat Moreover, non-steroidal aromatase inhibitors would not mammary carcinoma and Steele and colleagues have be expected to induce endometrial carcinoma in women shown that fadrozole completely inhibits the development and so could be investigated both as adjuvant hormonal of spontaneous breast tumors in aging Sprague-Dawley therapy as well as in the chemoprevention of human breast rats (Gunson et al. 1995). cancer. A few clinical studies have attempted to combine To assess whether aromatase inhibitors are superior to different classes of endocrine agents but there are few antiestrogens in the prevention of breast cancer, the clinical data to support this approach as being superior to optimal study would include patients at high risk of using these agents in sequence to treat metastatic breast developing breast cancer. Women with a single breast cancer, e.g. tamoxifen followed by an aromatase inhibitor, cancer are at high risk of developing a contralateral second followed by a progestin. In clinical practice, the sequential cancer. Estimates range from rates of 0.5 to 1.0% of use of hormonal agents can produce long-term palliation women per year for development of a contralateral breast of hormone-dependent breast cancer. Eventually, cancer. For a 60-year-old woman, this rate is 1.5- to 3.0- however, the problem of hormone resistance is encount- fold higher than the average incidence of 1:243 women per ered. The mechanism by which tumors become resistant year who develop their first primary tumor. Thus the to hormones in general are only partially understood. ATAC trial with assessment of diagnosis of second Refractoriness to therapy with aromatase inhibitors is primary tumors provides a powerful means of determining related not to the failure of these agents to suppress whether the aromatase inhibitors will prevent breast estradiol levels as might be seen if there were up- cancer. It is know that tamoxifen reduces the incidence of regulation of aromatase, but rather is likely due to some second primaries by 45% under these circumstances. other mechanism of hormone resistance. While trials of primary prevention of breast cancer with The paracrine production of aromatase-specific aromatase inhibitors are being planned, one would expect growth factors and cytokines within the microenviron- results from the adjuvant trials to be forthcoming sooner.

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